This invention relates to luminescent reagents having utility for monitoring specific binding reactions and, more particularly, to a luminol preparation having enhanced light output and stability.
Specific binding assays provide an economical means for detecting and measuring an analyte present in low concentrations in a sample. Specific binding assays are based upon the interaction of two bindable substances, one the analyte and the other a specific binding partner, which specifically recognize each other. Examples of specific binding partners whose interaction can serve as the basis for a specific binding assay include antigens-antibodies, biotin-avidin, nucleic acid probes, enzymes-substrates, enzymes-inhibitors, enzymes-cofactors, chelators-chelates, and cell surface receptor pairs. Assays involving other specifically bindable substances are also known and within the scope of the present invention. Specific binding assays have shown great utility in determining various analytes in biological, medical, environmental, agriculture and industrial applications.
A variety of assays using the principles of the specific binding approach are known, and several have become important diagnostic tools. In one such type of specific binding assay, the immunoassay, the analyte is an antibody, antigen, or hapten, and is made to react with another member of this group. While the background discussion will focus on such immunoassays, this focus is made for clarity of presentation, and is not to be interpreted as limiting of the invention, which is broadly applicable to luminescently labelled specific binding assays.
A variety of labelling reactions have been proposed for use in specific binding assays, including radioactive, enzymatic, chromogenic and luminogenic procedures. In a radioactive labelling procedure, the component conjugated with the specific binding partner is an atom or molecule which emits radioactivity. Chromogenic and luminogenic labelling reactions are chemically more complex, in that several reactants may be involved. The chromophore or lumiphore may itself be the label in the reaction, or a catalyst, typically an enzyme, may be used as the label. When the catalyst is used as the label, it will react with catalytic substrates which in turn produce color or luminescence. The remaining components of the reaction, that is, those not conjugated to the binding partner, are supplied in a chromogenic or luminogenic reagent medium, so that the uniting of the labelled conjugate and the reagent medium results in the desired color change or light emission, respectively.
Luminescent labels are attractive alternatives for use in specific binding assays for a variety of reasons. Luminescence is broadly defined as the production of visible light by atoms that have been excited by the energy produced in a chemical reaction, usually without an associated production of heat. Chemical energy excited electrons in the light-emitting molecules to higher energy states, from which electrons eventually fall to lower energy states with the emission of quanta of energy in the form of visible light. Luminescence is observed in several synthetic chemical compounds and also in naturally occurring biological compounds such as found in fireflies and certain species of fish.
One of the most important families of chemiluminescent molecules are the phthalylhydrazides. The most familiar member of this family is luminol, or 5-amino-2,3-dihydro-1,4-phthalazinedione, which has a gross chemical composition of C.sub.8 H.sub.7 N.sub.3 O.sub.2 and a double ring structure with a melting point of about 320.degree. C. Luminol is commercially available from several suppliers and is well characterized. Certain luminol analogs are also chemiluminescent, such as those wherein the position of the amino group is shifted (e.g., isoluminol, the amino group being at the 6 position), or is replaced by other substituents, as well as annelated derivatives and those with substitution in the nonheterocyclic ring. Some luminol analogs produce light more efficiently than does luminol itself, while others have lower efficiency. (As used herein, the term "luminol" encompasses such related species.)
Generally, luminol produces light in an oxidizing reaction, wherein the luminol combines with oxygen or an oxidizer to produce a reaction product and photons at a wavelength of about 425-450 nanometers (nm). The precise reaction formula and the quantum efficiency of light production (that is, the ratio of luminescing molecules to total molecules of the luminescent species) depend upon the medium in which the luminol resides, temperature and other reaction conditions. Typical oxidizers used in conjunction with luminol include oxygen, hydrogen peroxide, hypochloride, iodine, and permanganate.
The oxidation of luminol with the associated production of light occurs rather slowly at ambient temperatures, unless the reaction is catalyzed. A variety of different substances can catalyze the reaction, including organic enzymes (for example, horseradish peroxidase), other organic molecules such as microperoxidase and heme, positive metallic ions such as the cupric ion, and negative ions such as the ferricyanate ion.
Luminescent molecules would appear to be highly desirable as tags in specific binding assays because of their stability, sensitivity, the potential ease of detecting their emitted visible light and their lack of toxicity. Commercial luminol, however, has proven to be unsuitable for such purposes. There exists a need for specific improvements in the light emission characteristics of the reaction for use with such assays. Heretofore, commercial luminol has not shown sufficient activity to be useful to measure analytes at low concentrations in specific binding assays. The light emission intensity of the luminol reaction may be sufficient where high concentrations of catalyst are employed and where highly sophisticated and sensitive photometers are available, but the luminescent intensity has not been sufficient with low concentrations of catalyst and where other detection media such as photographic film or less sensitive photometers are used.
While the luminol reaction therefore offers important potential benefits in the measurement of the presence and amount of a reaction component, for many potential applications the intensity of the emitted light is too low. Further, the light emitted from commercial luminol exhibits an early flash of light without the first few seconds of the initiation of the reaction, followed by a progressive and rapid decrease in light emission over time. The integrated light intensity during any fixed period of time is therefore likely to be different from that measured over any other equal period of time. This variability may result in irreproducibility between tests. Desirably, there would be some period of time during which the light emission from the luminol reaction is relatively constant, so that the measurement of integrated light intensity could begin at different times after initiation of the reaction but within the period of constant light output, without variability of the results. This would eliminate the requirement that the reagents be added to a solution fixed in front of the luminescence detector which puts severe constraints on the light measuring system.
Inhibitors for the catalysts used in luminescent reactions have been reported (Theorell, The Enzymes, Vol. II, Part I, p. 397, Academic Press (1951)). However, the need to rigorously remove these inhibitors from luminogenic substrates has heretofore not been appreciated. We have discovered that rigorous removal of inhibitors from the luminescent substrates produces substantial improvement in the resulting sensitivity and reproducibility in specific binding assays. This is especially important when measuring low concentrations of analyte which necessitates low concentrations of catalyst.
There therefore exists a need for an improved luminescent substrate for use in specific binding assays and other applications. The improved luminescent substrate should exhibit an increased unit light intensity output per molecule of reacting luminescent substrate to allow its use as a tag in specific binding assays such as immunoassays where the concentration of analyte is low. The improved luminescent substrate should exhibit a period of substantially constant light output that would allow repetition of test procedures and eliminate many constraints on the user and the light detection system. The present invention fulfills these needs, and further provides related advantages.